奶牛安氏隐孢子虫巢氏PCR检测方法的建立及应用  

Establishment and Use of Nested PCR for Detection of Cryptosporidiosis andersoni in Dairy Cows

作　　者：张园园 张百慧 李简 刘书婷 韩莹 王燕春 蔡亚南[1] ZHANG Yuan-yuan;ZHANG Bai-hui;LI Jian;LIU Shu-ting;HAN Ying;WANG Yan-chun;CAI Ya-nan(College of Veterinary Medicine,Jilin Agricultural University,Changchun 130118,China)

机构地区：[1]吉林农业大学动物医学院,吉林长春130118

出　　处：《中国兽医杂志》2021年第12期28-32,共5页Chinese Journal of Veterinary Medicine

基　　金：国家重点研发计划项目(2018YFD0501600)。

摘　　要：为了建立敏感、稳定、重复性好的奶牛安氏隐孢子虫的巢氏PCR检测方法,本试验根据GenBank中安氏隐孢子虫18S rRNA和囊壁蛋白(COWP)基因序列设计2对引物,并通过程序优化建立了安氏隐孢子虫巢氏PCR检测方法。结果显示,18S rRNA和COWP巢氏PCR分别扩增出407 bp和430 bp的安氏隐孢子虫特异性基因片段,与预期相符,且扩增出的基因片段与已知基因序列(KX926454)同源性为100%;将阳性样本倍比稀释后进行18S rRNA和COWP巢氏PCR,最低检测量分别为24.3 pg/μL和243 pg/μL,扩增结果与目的片段大小一致,重复性良好,与大肠埃希菌、牛艾美尔球虫、弓形虫均无交叉反应;从华中地区采集的563份样品中,18S rRNA巢氏PCR检测出安氏隐孢子虫阳性185份,而COWP巢氏PCR仅检测出其中的148份;基于18S rRNA巢氏PCR,安氏隐孢子虫阳性率在秋季最高(36.71%),在冬季最低(16.25%),具有显著的统计学差异(P<0.01)。因此,本试验所建立的18S rRNA巢氏PCR和COWP巢氏PCR可为奶牛场安氏隐孢子虫的检测和防控提供有力的技术支持。This study aimed to establish and use sensitive,stable and reproducible nested PCR for detection of Cryptosporidium andersoni in dairy cows,with primers designed and optimized from C.andersoni 18S rRNA and oocyst wall protein(COWP)gene sequences.The results showed that the 18S rRNA and COWP nested PCR generated specific 407bp and 430bp fragments from C.andersoni 18S rRNA and COWP genes,respectively,with 100%homology to the known gene sequence(KX926454).The 18S rRNA and COWP nested PCR had limits of detection at 24.3 pg/L and 243 pg/L,respectively,yielded consistent target fragment sizes,and displayed no cross-reaction with Escherichia coli,Coccidioides niuemel and Toxoplasma gondii.Among the 185(32.86%)positives out of the 563 samples collected in central China by 18S rRNA nested PCR,148 were confirmed by the COWP nested PCR.Based on the 18S rRNA nested PCR,C.anderseni had the highest positive rate(36.71%)in autumn and the lowest positive rate(16.25%)in winter,with statistically significant difference(P<0.01).Thus,the 18S rRNA nested PCR and the COWP nested PCR described in this study offer a valuable tool for the detection,prevention and control of C.anderseni in dairy farms.

关 键 词：安氏隐孢子虫 巢氏PCR 18S rRNA 囊壁蛋白 检测 应用 

分 类 号：S852.723[农业科学—基础兽医学]